Microbes have been developed for use as vaccines that deliver heterologous antigens. Heterologous antigen delivery is provided by microbes that have been modified to contain nucleic acid sequences encoding a protein or antigen originating from a different species. Heterologous antigen delivery is especially advantageous for treating or preventing diseases or conditions that result from especially virulent or lethal sources, such as cancer and pathogenic agents (for example, HIV or Hepatitis B). Injection of a native or virulent infectious agent is potentially deleterious to the recipient organism. Likewise, a cancer cell which arises sporadically in an affected individual can subsequently propagate and likewise be potentially deleterious to a recipient organism. Heterologous antigen delivery is also especially advantageous where administration of attenuated or killed agent or cell has proven unsuccessful in eliciting an effective immune response or where sufficient attenuation of the infectious agent or cancer cell cannot be assured with acceptable certainty. Recently, certain bacterial strains have been developed as recombinant vaccines. For instance, an oral vaccine of attenuated Salmonella modified to express Plasmodium berghei circumsporozite antigen has been shown to protect mice against malaria (Aggarwal et al. 1990. J. Exp. Med. 172:1083).
One class of bacteria that can potentially be used as heterologous vaccines is facultative intracellular bacteria. The immune response to these bacteria can be a humoral response, a cell-mediated response, or both. However, killed intracellular bacteria or components of intracellular bacteria may not elicit a full cell-mediated immune response (Lauvau et el. 2001. Science 294:1735-9). These bacteria can spend a portion of their life cycle free in the circulatory or lymphatic systems of their host, where they are subject to the innate and antibody (i.e., humoral) responses of the host's immune system.
Facultative intracellular bacteria also may spend a portion of their life cycle sequestered within the host's cells, where they may be protected from the innate and humoral aspects of the host's immune system and may be susceptible to the cell-mediated responses of the host's immune system. A cell-mediated immune response is an immune response that stimulates effector T lymphocytes, which may in turn become memory (effector or central) T cells. A cell-mediated immune response results from the presentation of antigens on the surface of host cells. Phagocytic cells of the host's immune system can engulf live bacteria, killed bacteria or components of the bacteria into lysosomes, which mature into phagolysosomes and degrade protein antigens into peptides. Peptides of antigens contained within phagolysosomes of phagocytic cells may be presented on the surface of these phagocytic cells by MHC class II molecules for recognition by CD4+ T cells and the activation of a T helper response. Peptides of antigens expressed in the cytosol of any cell in the body of a mammal may be presented on the surface of that cell by MHC class I molecules for recognition by CD8+ T cells and the activation of a cytotoxic T cell (CTL) response. However, killed intracellular bacteria or components of intracellular bacteria may not invade non-phagocytic cells or may not escape from the phagolysosome of a phagocytic cell into the cytosol, resulting in activation and maturation of phagocytic cells, for example macrophages and dendritic cells. Therefore, the antigens of killed intracellular bacteria or components of intracellular bacteria may not be available for direct MHC I presentation and may not activate a CTL response. The ability of intracellular bacteria to produce proteins within the phagolysosomes and/or cytosol of the host may be necessary in order to elicit a fully effective cell-mediated immune response.
Strains of Listeria monocytogenes have recently been developed as intracellular delivery vehicles of heterologous proteins providing delivery of antigens to the immune system to induce an immune response to clinical conditions that do not permit injection of the disease-causing agent, such as cancer (U.S. Pat. No. 6,051,237 Paterson; U.S. Pat. No. 6,565,852) and HIV (U.S. Pat. No. 5,830,702, Portnoy & Paterson). As a facultative intracellular bacterium, L. monocytogenes elicits both humoral and cell-mediated bacterial antigen-specific immune responses. Following entry of the Listeria into a cell of the host organism, the Listeria produces Listeria-specific proteins that enable it to escape from the phagolysosome of the engulfing host cell into the cytosol of that cell. In the cell, L. monocytogenes proliferates, expressing proteins necessary for survival, but also expressing heterologous genes operably linked to Listeria promoters. Presentation of peptides of these heterologous proteins on the surface of the engulfing cell by MHC proteins permit the development of a T cell response. Since L. monocytogenes is a Gram-positive, food-borne human and animal pathogen responsible for serious infections in immunocompromised individuals and pregnant women, strains of these bacteria must be attenuated in a manner that reduces toxicity to the host, while maintaining immunogenicity of the vaccine. This toxicity is the result of bacterial invasion of various organs and tissues of the host, such as those of the liver, spleen and central nervous system. It would be beneficial to reduce the risks associated with using Listeria monocytogenes as a vaccine without affecting its potency to induce adaptive cell-mediated immunity specific for heterologous encoded antigen related to selected infectious and malignant diseases.